Abstract
Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer’s, Huntington’s, Parkinson’s diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons.
Highlights
Optimal brain function requires that neurons respond appropriately to a range of environmental challenges
Treatment with different concentrations of MG132, a proteasome inhibitor known to stabilize NMNAT214,20,35, increased the NMNAT2 Meso Scale Discovery (MSD) signal in a dose-dependent fashion up to 10 μM (Fig. 2A). Together these results suggest that this NMNAT2-MSD assay format detects endogenous NMNAT2 in cortical neurons with great specificity and sensitivity
We found that pretreatment with L-Aspartic acid, caffeine, and rolipram significantly reduced the impact of vincristine on the cell viability of NMNAT2 WT and HET, but not KO neurons (Fig. 6), while PD-169316 pre-treatment reduced vincristine toxicity in all genotypes
Summary
Optimal brain function requires that neurons respond appropriately to a range of environmental challenges. Recent studies have revealed a key neuronal maintenance and protective function for nicotinamide mononucleotide adenylyl transferases (NMNATs)[1,2]. Supporting its role as a potential neuronal/axonal maintenance factor, Nmnat[2] mRNA expression is reduced in many neurodegenerative diseases[24,25,26,27,28,29]. Our previous studies found NMNAT2 levels in Alzheimer’s disease (AD) brains are about 30% of control brains[6]. NMNAT2 levels in the brains from Non-Demented with Alzheimer’s disease. To identify small molecules that can modulate NMNAT2 levels, we developed a high-throughput screening platform to detect endogenous NMNAT2 levels in cortical neurons with high fidelity and great dynamic range. The nature of the small molecules identified to regulate NMNAT2 abundance suggests several upstream pathways modulate NMNAT2 levels in cortical neurons. We believe the knowledge of these pathways may promote better translational approaches for targeting NMNAT2 in various neurodegenerative diseases
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